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Overath, P., Weigel, U., Newhaus, J. M., Soppa, J., Seckler, R., Riede, I., Bocklage, H., Muller-Hill, B., Aichele, G., & Wright, J. K. (1987) Proc. Natl. Acad. Sci. U.S.A.84,5535. Padan, E., Sarkar, H. K., Viitanen, P. V., Poonian, M. S., & Kaback, H. R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765. Page, M . G. P., & Rosenbusch, J. P. (1988) J . Biol. Chem. 263, 15906. Piittner, I . B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. ( 1 986) Biochemistry 25, 4483. Piittner, I. B., Sarkar, H. K., Padan, E., Lolkema, J. S., & Kaback, H. R. ( 1 989) Biochemistry 28, 2525. Rees, C. C., DeAntonio, L., & Eisenberg, D. (1989) Science 245, 5 IO. Roepe, P. D., & Kaback, H. R. (1989) Biochemistry 28,6127. Roepe, P. D., Zbar, R., Sarkar, H. K., & Kaback, H. R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3992. Rudnick, G., Schuldiner, S., & Kaback, H. R. (1976) Biochemistry 25, 5 126. Sanger, F., & Coulson, A. R. (1978) FEBS Lett. 87, 107. Sanger, F., Nicklen, S., & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463. Sarkar, H. K., Viitanen, P. V., Padan, E., Trumble, W. R., Poonian, M. S., McComas, W., & Kaback, H. R. (1986) Methods Enzymol. 125, 21 4.
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Involvement of the Protein-Protein Interactions in the Thermodynamics of the Electron-Transfer Process in the Reaction Centers from Rhodopseudomonas viridis Laura Baciou,$ Tadeus Gulik-Krzywicki,s and Pierre Sebban*.$ U P R 407, Bat. 24, CNRS. GiflYuette 91198, France, and Centre de GPnCtique MolPculaire. GiflYuette 91198, France Received April 10, 1990; Revised Manuscript Received October 19, 1990
ABSTRACT: Reaction centers from Rhodopseudomonas viridis were reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine(DEPC) liposomes. Freeze-fracture electron micrographs were performed on the samples frozen from temperatures above and below the phase transition temperatures of those lipids (T, = 23 and 9.5 OC,in D M P C and DEPC, respectively). Above T,, in the fluid conformation of the lipids, the reaction centers are randomly distributed in the vesicle membranes. Below T,, aggregation of the proteins occurs. The Arrhenius plots of the rate constants of the charge recombination between P+ and QA- display a break a t about 24 OC in D M P C vesicles and about 10 O C in DEPC vesicles ( P represents the primary electron donor, a dimer of bacteriochlorophyll, and QA the primary quinone electron acceptor). This is in contrast to what was previously observed for the proteoliposomes of egg yolk phosphatidylcholine and for chromatophores [Baciou, L., Rivas, E., & Sebban, P. (1990) Biochemistry 29, 2966-29761, for which Arrhenius plots were linear. In D M P C and DEPC proteoliposomes, the activation parameters were very different on the two sides of T, (AH' for T < T, = 2.5 times AHo for T > Tc),leading however, to the same AGO values. Taking into account the structural and thermodynamic data, we suggest that, in vivo, protein-protein interactions play a role in the thermodynamic parameters associated with the energy stabilization process within the reaction centers.
x e light excitation energy harvested by the antenna of the photosynthetic organisms is converted at the level of the reaction centers into chemical free energy. This occurs via a transmembrane charge separation. In bacteria, the chroma'CNRS. 5Ccntrc dc GCnitique Moliculaire.
tophore membrane is mainly composed by phospholipids, 25% of total lipids being phosphatidylcholine (Niederman & Gibson, 1978; Rivas et al., 1987). The different kinetic steps of the electron transfer within the reaction centers as well as the prosthetic groups involved in these processes have been known for about 15 years. However, a main step for a better understanding of the energy stabilization in the reaction centers
0006-2960/9 1/0430- 1298$02.50/0 0 199 1 American Chemical Society
Biochemistry, Vol. 30, No. 5, I991
Electron Transfer in Rps. viridis Reaction Centers has been accomplished with the crystallization and X-ray structural analysis of the reaction center protein from the purple bacteria Rhodopseudomonas uiridis (Deisenhofer & al., 1985; Michel & Deisenhofer, 1988; Michel & al., 1986) and Rhodobacter sphaeroides (Allen et al., 1988; Chang et al., 1986; Ducruix & Reiss-Husson, 1987; Komiya et al., 1988; Yeates et al., 1988). The reaction center consists of three polypeptides, L, M, and H, which molecular weights range between 30 and 35 kDa. In Rps. ciridis, a tightly bound cytochromc (40 kDa) containing four C-type hemes is found (Weyer et al., 1987). The L and M polypeptides are almost completcly embedded in the chromatophore membrane, whereas H and the cytochrome, which are more hydrophilic, are situated on the cytoplasmic and periplasmic sides of the membrane, respectively. The pigments involved in the electron-transfer process are located on the L and M subunits. The primary electron donor is a dimer of bacteriochlorophylls (P), which, in its electronic excited state P*, transfers an electron to a bacteriopheophytin (1) in less than 5 ps, possibly via a monomeric bacteriochlorophyll (Holzapfel et al., 1990). The P+I- state decays in about 200 ps by electron donation to the primary quinone molecule, Q A . Under normal conditions, the electron prcscnt on Q A is transferred to a secondary quinone molecule QB in 20-200 p s , depending on the bacterial species. However, in the presence of electron-transfer inhibitors that compete with Qe for its site, the P'QA- state decays by recombination in about 1.5 ms a t pH 8 in Rps. viridis (Baciou et al., 1990; Sebban & Wraight, 1989; Shopes & Wraight, 1987). Thc decay kinetics associated with this process are biphasic (Baciou et al., 1990; Sebban & Wraight, 1989). Because they were observed in chromatophores, and also on the P'QB- charge recombination, which decays in about 500 ms at pH 7, these phases were attributed to two conformational states of the reaction centers, preexisting in the dark (Baciou et al., 1990). In bacterial strains such as Rps. viridis, where the energy gap between the initial state P* and P'QA- is smaller than 0.8 eV (Gunner et al., 1986), P'QA- recombines via a relaxcd state of P'I- (Kleinfeld et al., 1985; Gopher et al., 1985; Gunner et al., 1986; Sebban, 1988; Sebban & Wraight, 1989; Shopes & Wraight, 1987; Woodbury et al., 1986). Therefore the Arrhenius plots of the rate constant of P+QA-charge recombination were found linear in reaction centers isolated in detergent (Sebban & Wraight, 1989; Shopes & Wraight, 1987), reconstituted in phosphatidylcholine (egg yolk) (PC)I vesicles and in chromatophores (Baciou et al., 1990). Little is known about the reaction center-lipid interactions and about the influence of lipids on the electrontransfer processes within the reaction centers. In this work we have reconstituted reaction centers from Rps. uiridis into dimyristoylphosphatidylcholine (DMPC) and in dielaidoylphosphatidylcholine (DEPC) liposomes. We show that the lipid phase transition does affect the thermodynamics parameters associated with the electron-transfer process in the reaction centers. Moreover, in the view of the freeze-fracture electron micrographs we suggest that protein-protein interactions play a role in the energy stabilization function of the reaction centers, in vivo. MATERIAL A N D METHODS Wild-type Rps. viridis cells were grown anaerobically (N, and COz) in the light in the Hutner medium. Reaction centers were prepared as previously described (Prince & Dutton, I Abbreviations: D M P C . dimyristoylphosphatidylcholine: DEPC, dielaidoylphosphatidylcholine;LDAO, lauroyldimethylamine N-oxide; PC, phosphatidylcholinc.
1299
1976). DMPC (14:0/14:0) was obtained from Bachem and DEPC (18:ltA9/18:ltA9)from Avanti. The DMPC proteoliposomes were prepared by using the same method as previously described for PC proteoliposomes, except that the liposome preparation, the reconstitution with the reaction centers, and the elimination of detergent (LDAO) on a Sepharose CL-4B column were all done at about 26 OC for DMPC and about 20 OC for DEPC, i.e., above the phase transition temperatures ( T c ) .The lipid to protein ratio was 3:l(w/w). For freeze-fracture electron microscopy, small drops of about 50 pL containing 25-30% glycerol were deposited on conventional Balzers gold planchets and rapidly frozen (about 100000 O / s ) in Freon 22 at -160 OC. Fracturing and replication were done with Balzers BAF 301 freeze-etching unit by using platinum-carbon shadowing. The replicas, after digestion of organic material with chromic acid and washing with distilled water, were observed in a Philips 301 electron microscope. The flash absorption spectroscopy apparatus was the same as previously described (Baciou et al., 1990). The temperature was monitored by using a NiCr-Ni thermometer with a precision of f0.3 "C. For the activation energy measurements, pH was measured in line and readjusted at different temperatures. RESULTS The P'QA- charge recombination kinetics observed in DMPC and DEPC proteoliposomes were biphasic, as was already mentioned for the reaction centers in detergent (Sebban & Wraight, 1989), in P C liposomes, and in chromatophores (Baciou et al., 1990). In DMPC liposomes, a t 298 K, the two lifetimes, measured a t pH 9, a t 960 nm, are equal to l/kfaSl= 0.72 f 0.05 ms (50%) and I/kslow= 2.48 f 0.05 (50%), in good agreement with our previous data on PC liposomes, for which the lifetimes were 0.68 and 2.2 ms, respectively. The Arrhenius plots of kfasland k,,,, are represented in Figures 1 and 2 for DMPC and DEPC, respectively. It was previously suggested that, in Rps. viridis, at room temperature, charge recombination occurs by a thermally activated process (Shopes & Wraight, 1987). The state via which P'QA- recombines was suggested to be a relaxed state of P'I- (Kleinfeld et al., 1985; Gopher et al., 1985; Gunner et al., 1986; Sebban, 1988; Sebban & Wraight, 1989; Shopes & Wraight, 1987; Woodbury et al., 1986). Because the rate of electron transfer from P+I- to P'QA- is much higher than the deactivation rates from either P'I- or P'QA-, it was postulated that thermal equilibrium is established between these states. In other words it was assumed that, for the two components (Sebban & Wraight, 1989) kslow
kfast
=
kd
exp(-Acoslow/kBT)
+ kTslow
=
kd
exp(-AcOfas:/kBT)
+
kTfas:
(1)
where kd is the rate constant of charge recombination from P'I-. We used kd = 2 X lo7 s-' (Holten et al., 1978; Shopes & Wraight, 1987). An inherent hypothesis to eq 1 is that kd is the same for the fast and the slow component. This is probably correct since it was reported by Woodbury and Parson (1984) that the decay of the absorption changes associated with P'1- is fitted by a single exponential (C. E. D. Chidsey, C. Kirmaier, D. Holten, and S. G. Boxer, personal communication; A. C. van Bochove, R. van Grondelle, N. Woodbury, and W. W . Parson, unpublished data). ACofast and AGo,,,, are the free energy differences between P'I- and P+QA-for the fast and the slow phase, respectively. At low
Baciou et al.
1300 Biochemistry, Vol. 30, No. 5, 1991 7.2
8 Ln (k fast- kTfast)
7.0 76.8
6.6 6 6.4
..\
i
5 -
1
I
'
1
'
1
'
1
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'
.\
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Arrhenius plots of the PQAcharge recombination kinetics in Rps. oiridis reaction centers reconstituted in DMPC vesicles. kTf& and kTslavwere taken equal to 150 and 600 8,respectively (see text).
Arrhenius plots of the PQAcharge recombination kinetics in Rps. uiridis reaction centers reconstituted in DEPC vesicles. kTf& and kTSI, were taken equal to 150 and 600 s-l, respectively (see text).
temperature (Tc, frozen from 30 OC,
